Mapping and sequencing of the dihydrofolate reductase gene ( DFR1) of Saccharomyces cerevisiae

• Published in: Gene Vol. 63; no. 2; pp. 175 - 185
• Main Authors: Barclay, Barry J., Huang, Tun, Nagel, Michael G., Misener, Virginia L., Game, John C., Wahl, Geoffrey M.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 31.03.1988; Amsterdam Elsevier; New York, NY
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; Biotechnology; Cloning, Molecular; codon usage; DNA Restriction Enzymes; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; gene cloning; Genes; Genes, Fungal; Genes. Genome; Genetic engineering; Genetic technics; homology; Methods. Procedures. Technologies; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Nucleic Acid Hybridization; Plasmids; Recombinant DNA; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Synthetic digonucleotides and genes. Sequencing; Tetrahydrofolate Dehydrogenase - genetics; transcriptional control elements; trimethoprim and methotrexate resistance; NADPH; gene cloning; homology; DHF; transcriptional control elements; nt; UAS; trimethoprim and methotrexate resistance; Cm; Recombinant DNA; Chr; codon usage; bp; Ap; Mtx; R; THF; kb; Tp; aa; DFR1 (S. cerevisiae); DHFR; dUMP; dTMP; TS; Yeast; Nucleotide sequence; Enzyme; Tetrahydrofolate dehydrogenase; Fungi; Resistance; Gene; Restriction map; Regulatory sequence; Ascomycetes; Molecular cloning; Saccharomyces cerevisiae; Thallophyta
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(88)90523-9

The dihydrofolate reductase gene ( DFR1) from Saccharomyces cerevisiae has been mapped and sequenced. The gene was isolated on an 8.8-kb BamHI fragment from a yeast genomic library by screening of Escherichia coli transformants for resistance to trimethoprim. A 1.8-kb SalI- BamHI fragment which was able to confer methotrexate resistance in yeast also complemented an E. coli DHFR-deficient ( folA) mutant. Nucleotide sequence analysis revealed that the yeast DFR1 gene encoded a polypeptide with a predicted M r of 24230. The deduced sequence of 211 amino acid residues showed considerable homology with DHFRs from both bacterial and animal sources. The codon bias index of the DFR1 coding region is 0.0083, which indicates a random pattern of codon usage. The upstream region contains two consensus sequences required for binding of the yeast's positive regulatory factor, GCN4, suggesting that the DFR1 gene might be subject to the amino acid general control. Several potential ‘TATA’ boxes are located in the sequence 5' to the gene. Located in the 3' flanking region are homologies with several canonical sequences thought to be required for efficient transcription termination in yeast. We also mapped the DFR1 gene to a position 1.4 cM proximal to the MET7 locus on chromosome XV.